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ObjectivesPrevotella copriis considered to be a contributing factor in rheumatoid arthritis (RA). However, in some non-Westernised countries, healthy individuals also harbour an abundance ofP. copriin the intestine. This study investigated the pathogenicity of RA patient-derivedP. copri(P. copriRA) compared with healthy control-derivedP. copri(P. copriHC).MethodsWe obtained 13P.copristrains from the faeces of patients with RA and healthy controls. Following whole genome sequencing, the sequences ofP. copriRAandP. copriHCwere compared. To analyse the arthritis-inducing ability ofP. copri, we examined two arthritis models (1) a collagen-induced arthritis model harbouringP. copriunder specific-pathogen-free conditions and (2) an SKG mouse arthritis model underP. copri-monocolonised conditions. Finally, to evaluate the ability ofP. coprito activate innate immune cells, we performed in vitro stimulation of bone marrow-derived dendritic cells (BMDCs) byP. copriRAandP. copriHC.ResultsComparative genomic analysis revealed no apparent differences in the core gene contents betweenP. copriRAandP. copriHC, but pangenome analysis revealed the high genome plasticity ofP. copri. We identified aP. copriRA-specific genomic region as a conjugative transposon. In both arthritis models,P. copriRA-induced more severe arthritis thanP. copriHC. In vitro BMDC stimulation experiments revealed the upregulation of IL-17 and Th17-related cytokines (IL-6, IL-23) byP. copriRA.ConclusionOur findings reveal the genetic diversity ofP. copri, and the genomic signatures associated with strong arthritis-inducing ability ofP. copriRA. Our study contributes towards elucidation of the complex pathogenesis of RA.

ObjectiveGenome-wide association studies (GWAS) have identified >100 risk loci for systemic lupus erythematosus (SLE), but the disease genes at most loci remain unclear, hampering translation of these genetic discoveries. We aimed to prioritise genes underlying the 110 SLE loci that were identified in the latest East Asian GWAS meta-analysis.MethodsWe built gene expression predictive models in blood B cells, CD4+and CD8+T cells, monocytes, natural killer cells and peripheral blood cells of 105 Japanese individuals. We performed a transcriptome-wide association study (TWAS) using data from the latest genome-wide association meta-analysis of 208 370 East Asians and searched for candidate genes using TWAS and three data-driven computational approaches.ResultsTWAS identified 171 genes for SLE (p–5); 114 (66.7%) showed significance only in a single cell type; 127 (74.3%) were in SLE GWAS loci. TWAS identified a strong association betweenCD83and SLE (p–8). Meta-analysis of genetic associations in the existing 208 370 East Asian and additional 1498 cases and 3330 controls found a novel single-variant association at rs72836542 (OR=1.11, p=4.5×10–9) aroundCD83. For the 110 SLE loci, we identified 276 gene candidates, including 104 genes at recently-identified SLE novel loci. We demonstrated in vitro that putative causal variant rs61759532 exhibited an allele-specific regulatory effect onACAP1, and that presence of the SLE risk allele decreasedACAP1expression.ConclusionsCell-level TWAS in six types of immune cells complemented SLE gene discovery and guided the identification of novel genetic associations. The gene findings shed biological insights into SLE genetic associations.

BackgroundKrebs von den Lungen-6 (KL-6) is a known biomarker for diagnosis and monitoring of interstitial lung diseases. However, the role of serum KL-6 and the mucin 1 (MUC1) variant (rs4072037) in COVID-19 outcomes remains to be elucidated. We aimed to evaluate the relationships among serum KL-6 levels, critical outcomes and theMUC1variant in Japanese patients with COVID-19.MethodsThis is a secondary analysis of a multicentre retrospective study using data from the Japan COVID-19 Task Force collected from February 2020 to November 2021, including 2226 patients with COVID-19 whose serum KL-6 levels were measured. An optimal serum KL-6 level cut-off to predict critical outcomes was determined and used for multivariable logistic regression analysis. Furthermore, the relationship among the allele dosage of theMUC1variant, calculated from single nucleotide polymorphism typing data of genome-wide association studies using the imputation method, serum KL-6 levels and COVID-19 critical outcomes was evaluated.ResultsSerum KL-6 levels were significantly higher in patients with COVID-19 with critical outcomes (511±442 U/mL) than those without (279±204 U/mL) (pMUC1variant was independently associated with increased serum KL-6 levels (aOR 0.24, 95% CI 0.28 to 0.32) but not significantly associated with critical outcomes (aOR 1.11, 95% CI 0.80 to 1.54).ConclusionSerum KL-6 levels predicted critical outcomes in Japanese patients with COVID-19 and were associated with theMUC1variant. Therefore, serum KL-6 level is a potentially useful biomarker of critical COVID-19 outcomes.

ObjectiveSystemic lupus erythematosus (SLE), an autoimmune disorder, has been associated with nearly 100 susceptibility loci. Nevertheless, these loci only partially explain SLE heritability and their putative causal variants are rarely prioritised, which make challenging to elucidate disease biology. To detect new SLE loci and causal variants, we performed the largest genome-wide meta-analysis for SLE in East Asian populations.MethodsWe newly genotyped 10 029 SLE cases and 180 167 controls and subsequently meta-analysed them jointly with 3348 SLE cases and 14 826 controls from published studies in East Asians. We further applied a Bayesian statistical approach to localise the putative causal variants for SLE associations.ResultsWe identified 113 genetic regions including 46 novel loci at genome-wide significance (p−8). Conditional analysis detected 233 association signals within these loci, which suggest widespread allelic heterogeneity. We detected genome-wide associations at six new missense variants. Bayesian statistical fine-mapping analysis prioritised the putative causal variants to a small set of variants (95% credible set size ≤10) for 28 association signals. We identified 110 putative causal variants with posterior probabilities ≥0.1 for 57 SLE loci, among which we prioritised 10 most likely putative causal variants (posterior probability ≥0.8). Linkage disequilibrium score regression detected genetic correlations for SLE with albumin/globulin ratio (rg=−0.242) and non-albumin protein (rg=0.238).ConclusionThis study reiterates the power of large-scale genome-wide meta-analysis for novel genetic discovery. These findings shed light on genetic and biological understandings of SLE.

ObjectivesSystemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by autoantibody production and widespread inflammation damaging many organs. Previous genome-wide association studies (GWASs) have revealed over 80 genetic determinants of SLE, but they collectively explain a fraction of the heritability, and only a few were proven in vivo for the involvement in SLE. We conducted a meta-analysis of SLE GWAS in the Japanese population, followed by functional analyses of a susceptibility gene with use of mutant mice.MethodsWe conducted a meta-analysis of two GWASs comprising a total of 1363 cases and 5536 controls using the 1000 Genome Project data as an imputation reference. Enrichment analyses for functional annotations were conducted. We examined Phospholipase D4 (Pld4) mutant mice to assess functional involvement of a genetic determinant.ResultsWe found a total of 14 significant loci, which included rs2582511 in AHNAK2/PLD4 recently reported in a Chinese study and a novel locus of rs143181706 in MAMLD1 (p=7.9×10−11 and 3.7×10–8, respectively). PLD4 risk allele was associated with anti-dsDNA antibody production. Enrichment analysis of genetic signals revealed involvement of a wide range of immune-related cells and pathways. Pld4 mutant mice revealed remarkably low body weight. The mice demonstrated autoimmune phenotypes compatible with SLE, including splenomegaly and lymphadenopathy, expansion of B cells and hypersecretion of BAFF and production of autoantibodies especially anti-nuclear antibody and anti-dsDNA antibody.Conclusions We found a novel susceptibility gene to SLE. Pld4 mutant mice revealed autoimmune phenotypes suggesting functional involvement of PLD4 with the basics of SLE.

Study of the genetics of rheumatoid arthritis (RA) began about four decades ago with the discovery of HLA-DRB1. Since the beginning of this century, a number of non-HLA risk loci have been identified through genome-wide association studies (GWAS). We now know that over 100 loci are associated with RA risk. Because genetic information implies a clear causal relationship to the disease, research into the pathogenesis of RA should be promoted. However, only 20% of GWAS loci contain coding variants, with the remaining variants occurring in non-coding regions, and therefore, the majority of causal genes and causal variants remain to be identified. The use of epigenetic studies, high-resolution mapping of open chromatin, chromosomal conformation technologies and other approaches could identify many of the missing links between genetic risk variants and causal genetic components, thus expanding our understanding of RA genetics.

ObjectiveIn anti-citrullinated protein antibody positive rheumatoid arthritis (ACPA-positive RA), a particular subset of HLA-DRB1 alleles, called shared epitope (SE) alleles, is a highly influential genetic risk factor. Here, we investigated whether non-HLA single nucleotide polymorphisms (SNP), conferring low disease risk on their own, interact with SE alleles more frequently than expected by chance and if such genetic interactions influence the HLA-DRB1 SE effect concerning risk to ACPA-positive RA.MethodsWe computed the attributable proportion (AP) due to additive interaction at genome-wide level for two independent ACPA-positive RA cohorts: the Swedish epidemiological investigation of rheumatoid arthritis (EIRA) and the North American rheumatoid arthritis consortium (NARAC). Then, we tested for differences in the AP p value distributions observed for two groups of SNPs, non-associated and associated with disease. We also evaluated whether the SNPs in interaction with HLA-DRB1 were cis-eQTLs in the SE alleles context in peripheral blood mononuclear cells from patients with ACPA-positive RA (SE-eQTLs).ResultsWe found a strong enrichment of significant interactions (AP pHLA-DRB1 SE alleles and the group of SNPs associated with ACPA-positive RA in both cohorts (Kolmogorov-Smirnov test D=0.35 for EIRA and D=0.25 for NARAC, pHLA-DRB1 SE alleles for disease decreases from 5.2 to 2.5 after removal of the risk alleles of the two top interacting SNPs (rs2476601 and rs10739581).ConclusionOur data demonstrate that there are massive genetic interactions between the HLA-DRB1 SE alleles and non-HLA genetic variants in ACPA-positive RA.

Autoimmune diseases including rheumatic diseases affect 3%–10% of the world population. The frequency of incidence is dependent on the geographical area, ethnicity, sex and environmental factors.1 Except for rare monogenic diseases, the majority of autoimmune diseases are defined as a complex trait disorder, where multiple genetic and environmental factors interact. For example, twin studies have estimated that the heritability of rheumatoid arthritis (RA) is ~60%.2 3 RA is classified into anti-citrullinated protein antibodies (ACPA) positive and negative subtypes. The above-mentioned heritability of RA applies primarily to patients with RA who are positive for ACPAs, whereas the heritability of seronegative RA appears to be low,4 suggesting the differences in the genetic background of these two conditions. Therefore, although the classification criteria are satisfied, each autoimmune disease appears to be heterogeneous. On the other hand, autoimmune diseases share several symptoms, such as Raynaud phenomenon and arthritis. In a clinical situation, the terms ‘poly-autoimmunity’ and ‘familial autoimmunity’ are used. The former suggests the presence of multiple autoimmune diseases in a single patient, and the latter implies the situation where diverse autoimmune diseases are persistent in a family. Genes in the major histocompatibility complex (MHC) have been reported to be the strongest single genetic effect in the majority of autoimmune diseases. Although the precise mechanisms remain to be discovered, these MHC loci are the first and the most robust examples of shared genetic factors among several autoimmune diseases. Since 2007, rapid advances in genome-wide association study (GWAS) have enhanced the identification of hundreds of genetic risk factors for many complex diseases. Thereafter, one possible approach to expanding our understanding of the genetics of autoimmune disease using GWAS data is cross-trait analyses to identify genetic correlations with other complex traits. In fact, it has been reported that genetic variants associated with one …

Background HLA-DRB1 is the strongest susceptibility gene to rheumatoid arthritis (RA). HLA-DRB1 alleles showed significant non-additive and interactive effects on susceptibility to RA in the European population, but these effects on RA susceptibility should vary between populations due to the difference in allelic distribution. Furthermore, non-additive or interactive effects on the phenotypes of RA are not fully known. We evaluated the non-additive and interactive effects of HLA-DRB1 alleles on RA susceptibility and anticitrullinated protein/peptide antibody (ACPA) levels in Japanese patients. Methods A total of 5581 ACPA(+) RA and 19 170 controls were genotyped or imputed for HLA-DRB1 alleles. Logistic regression analysis was performed for both allelic non-additive effects and interactive effects of allelic combinations. The significant levels were set by Bonferroni’s correction. A total of 4371 ACPA(+) RA were analysed for ACPA levels. Results We obtained evidence of non-additive and interactive effects of HLA-DRB1 on ACPA(+) RA susceptibility (p=2.5×10 −5 and 1.5×10 −17 , respectively). Multiple HLA-DRB1 alleles including HLA-DRB1 *04:05, the most common susceptibility allele in the Japanese, showed significant non-additive effects (p≤0.0043). We identified multiple allelic combinations with significant interactive effects including a common combination with the European population as well as novel combinations. Additional variance of ACPA(+) RA susceptibility could be explained substantially by heterozygote dominance or interactive effects. We did not find evidence of non-additive and interactive effects on levels of ACPA. Conclusion HLA allelic non-additive and interactive effects on ACPA(+) RA susceptibility were observed in the Japanese population. The allelic non-additive and interactive effects depend on allelic distribution in populations.

We thank Liu et al for their interest in our paper and for providing their thoughts through correspondence.1 We agree that gut microbiome studies are promising to identify novel insights into the sexual dimorphism in rheumatoid arthritis (RA). We reported that the genus of Gardnerella and the species of Gardnerella vaginalis increased in the gut microbiome of patients with RA.2 G. vaginalis is known as a representative causal bacterium of vaginosis. In our study, G. vaginalis was detected in both male and female samples, and there were no significant gender differences in their relative abundance ( P =0.41). Thus, the increment of G. vaginalis was less likely to be contamination from the female genital organ in perineum. We further performed case-control association tests of G. vaginalis stratified by gender with age, sequencing groups and the top two principal components as covariates (figure 1A). We found that the effect size was larger in female samples …

Objectives: While genetic determinants of low density lipoprotein (LDL) cholesterol levels are well characterised in the general population, they are understudied in rheumatoid arthritis (RA). Our objective was to determine the association of established LDL and RA genetic alleles with LDL levels in RA cases compared with non-RA controls. Methods: Using data from electronic medical records, we linked validated RA cases and non-RA controls to discarded blood samples. For each individual, we extracted data on: first LDL measurement, age, gender and year of LDL measurement. We genotyped subjects for 11 LDL and 44 non-HLA RA alleles, and calculated RA and LDL genetic risk scores (GRS). We tested the association between each GRS and LDL level using multivariate linear regression models adjusted for age, gender, year of LDL measurement and RA status. Results: Among 567 RA cases and 979 controls, 80% were female and mean age at the first LDL measurement was 55 years. RA cases had significantly lower mean LDL levels than controls (117.2 vs 125.6 mg/dl, respectively, p, National Institutes of Health (U.S.) (Grant U54-LM008748)

Objectives:Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors, which regulate the production of proinflammatory cytokines. Polymorphisms in the IRF5 gene have been associated with susceptibility to systemic lupus erythaematosus (SLE) in Caucasian and Asian populations, but their involvement in other autoimmune diseases is still uncertain. Here, we assessed the genetic role of IRF5 in susceptibility to rheumatoid arthritis (RA) in Japanese subjects.Methods:We selected 13 single nucleotide polymorphisms (SNPs) and a CGGGG insertion–deletion polymorphism in the IRF5 gene. We performed 2 sets of case–control comparisons using Japanese subjects (first set: 830 patients with RA and 658 controls; second set: 1112 patients with RA and 940 controls), and then performed a stratified analysis using human leukocyte antigen (HLA)-DRB1 shared epitope (SE) status. We genotyped the SNPs using TaqMan assays.Results:A significant association of the rs729302 A allele with RA susceptibility was found in both sets (odds ratio (OR) 1.22, 95% CI 1.09 to 1.35, pConclusions:These findings indicate that the promoter polymorphism of IRF5 is a genetic factor conferring predisposition to RA, and that it contributes considerably to disease pathogenesis in patients that were SE negative.

Background Susceptibility genes for anti-citrullinated peptide/protein antibodies (ACPA)-positive rheumatoid arthritis (RA) have been successfully discovered by the development of genome-wide association studies (GWAS). However, little is known about the genetic background of ACPA-negative RA. Objectives We intended to clarify genetic background of ACPA-negative RA and compare susceptibility genes between ACPA-negative and -positive RA. Methods We performed a meta-analysis of GWAS comprising 670 ACPA-negative RA and 16,891 controls for 1,948,138 markers, followed by a replication study of the top 35 SNPs using 916 cases and 3,764 controls. Inverse-variance method was applied to assess overall effects. To assess overlap of susceptibility genes between ACPA-positive and -negative RA, odds ratios (ORs) of the 21 susceptibility markers to RA in Japanese were compared between the two subsets. In addition, SNPs were stratified by the p-values in GWAS meta-analysis for either ACPA-positive RA or ACPA-negative RA to address the question whether weakly-associated genes were also shared. The correlations between ACPA-positive RA and the subpopulations of ACPA-negative RA (rheumatoid factor (RF)-positive and RF-negative subsets) were also addressed. Results Rs6904716 in LEMD2 of the HLA locus showed a borderline association with ACPA-negative RA (overall p=5.7x10–8), followed by rs6986423 in CSMD1 (p=2.4x10–6) and rs17727339 in FCRL3 (p=1.4x10–5). ACPA-negative RA showed significant correlations of ORs with ACPA-positive RA for the 21 susceptibility SNPs and non-HLA SNPs with p-values far from significance. These significant correlations with ACPA-positive RA were true for ACPA-negative RF-positive and ACPA-negative RF-negative RA. On the contrary, positive correlations were not observed between the ACPA-negative two subpopulations. ![Figure][1] Conclusions Many of the susceptibility genes were shared between ACPA-positive and -negative RA. Acknowledgements We'd like to thank all doctors who helped us to collect DNA samples from patients with ACPA-negative RA. We'd like to thank especially Drs. Takao Fujii, Hiromu Ito, Motomu Hashimoto and Moritoshi Furu for their effort for sample accumulation. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.3219 [1]: pending:yes

Background Several studies have tested the hypothesis that RA susceptibility variants associate with radiological progression. Their findings are often not replicated; additionally they have examined only a proportion of risk loci and evaluated each locus separately, despite limited power. Two recent meta-analyses of genome-wide association studies have expanded the number of genetic loci and HLA amino acid polymorphisms associated with RA susceptibility [1, 2]. Our aim was to establish if these variants correlated with radiological progression in early active RA patients enrolled to a clinical trial. We evaluated their association both individually and cumulatively, using a weighted genetic risk score (wGRS) combining risk loci. Objectives To establish if genetic predictors of RA susceptibility also associate with radiological progression in early active RA. Methods We evaluated 421 RA patients previously enrolled to the Combination Anti-Rheumatic Drugs in Early RA (CARDERA) trial. Hand/feet radiographs were read for modified Larsen scores every 6 months for 2 years. We evaluated the association of validated genetic risk variants (69 SNPs, 17 four-digit HLA-DRB1 alleles and polymorphisms in 6 HLA amino acid positions) with log-transformed Larsen scores over time using a linear mixed-effects model. Log e (Larsen score + 1) was the response variable. Time, treatment, age, disease duration, ACPA status, genotype and a genotype*time interaction, were the fixed-effects predictor variables. Genotype was coded additively. All β-estimates were back-transformed to the original Larsen scale. The genotype*time β provided information on the annual increase in Larsen score per risk allele copy relative to the reference genotype; P -values for this term provided information on the variant9s role in radiological progression. We assessed the association between a wGRS incorporating susceptibility SNPs and radiological progression by replacing the linear model9s genotype term with a wGRS. Ethical approval was obtained; all patients provided consent. Results Two SNPs, one HLA-DRB1 allele and two HLA-DRβ1 protein amino acid polymorphisms had P -values P =0.0680). Conclusions RA susceptibility loci did not have a clinically relevant association with radiological progression in European ancestry, early, active RA patients recruited to this trial. This suggests the genetic architectures of RA susceptibility and severity differ. References Okada Y et al. Genetics of rheumatoid arthritis contributes to biology and drug discovery. Nature 2013; [Epub ahead of print]. Raychaudhuri S et al. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis. Nat Genet 2012;44:291-6. Acknowledgements Arthritis Research UK (Grant Reference Number 19739 to ICS); National Institute for Health Research (NIHR) Biomedical Research Centre at Guy9s and St Thomas9 NHS Foundation Trust in partnership with King9s College London. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5018

International audience; Objective To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the RA genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. Methods We analyzed a total of 11,715 RA cases and 26,493 controls from 9 independent cohorts, all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on OPN expression in macrophages and OPN serum levels was investigated. Results We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥ 3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (P = 1.29x10-5, ORACPA-negative 1.257 1.135–1.394) and less with ACPA positivity (P = 0.0148, ORACPA-positive 1.072 1.014–1.134]). The odds ratios between these subgroups (i.e., ACPA-positive and -negative) significantly differed (P = 7.33x10-3). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (P = 0.0157) as well as serum level of secreted OPN correlated positively with ACPA production (P = 0.005; r = 0.483). Conclusion We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association greater in patients negative for ACPAs.

Anti-cyclic citrullinated peptide (anti-CCP) antibody is a highly specific biomarker for rheumatoid arthritis (RA), and is recognised as a predictor of the development of RA.1 2 It has been demonstrated that some HLA-DRB1 alleles, such as shared epitope (SE) alleles, significantly contribute to the positivity of anti-CCP antibody and the susceptibility of anti-CCP antibody-positive RA.3 4 However, the quantitative effect of HLA-DRB1 alleles on anti-CCP antibody levels in RA patients is controversial.3 5 6 Therefore, we carried out a large-scale study to study the quantitative effects of HLA-DRB1 alleles on anti-CCP antibody levels in Japanese patients with RA. A total of 2384 Japanese patients with RA was recruited through several medical institutes of Japan under the support of the BioBank Japan Project7 (age 61.5±11.8 years (mean±SD), 81.2% were women). All patients fulfilled the American College of Rheumatology revised criteria for RA, and provided written informed consent. Serum anti-CCP antibody levels were assayed using the second-generation anti-CCP ELISA kit (DIASTAT Anti-CCP; MBL, Nagoya, Japan) with …